The present invention relates to the finding of Helicobacter pylori (H. pylori) antigens and antigenic fragments thereof in blood, which includes whole blood, plasma, and serum. The H. pylori antigens found in blood include, but not limited to, H. pylori DNA, RNA, or fragments thereof, or H. pylori proteins/peptides or other antigenic components thereof. H. pylori DNA or fragments thereof are detected by polymerase chain reaction (PCR), ligase chain reaction (LCR), DNA hybridization, branched DNA signal amplification assay, or other signal amplification methods. H. pylori RNA thereof are detected by PCR, hybridization or other signal amplification assays. H. pylori proteins or peptides or other antigenic components thereof are detected by immunoassays or immunoblotting using an affinity purified antibody against H. pylori. The present invention also relates to diagnosing H. pylori infection by detecting the H. pylori antigens in blood.
Helicobacter pylori (H. pylori) is a gram-negative bacterium which infects the gastric mucosal and is responsible for most peptic ulcer disease (PUD). Until recently, ulcers and other forms of dyspepsia were thought to be related to stress levels or eating habits. Recently, the medical community has confirmed that H. pylori is the causative agent for certain forms of gastric distress, including ulcers and gastric cancer. Eradication of H. pylori promotes healing of ulcer and greatly reduces the incidences of cancer and PUD.
H. pylori causes most gastric and duodenal ulcers, as well as peptic ulcer disease (PUD). The linkage of H. pylori and PUD was first discovered and published by Australian physicians Warren and Marshall in 1984 (Lancet I: 1311-1344). The H. pylori infection is now accepted as the most common cause of gastritis, and is etiologically involved in gastric ulcer, duodenal ulcer, gastric adenocarcinoma and primary B-cell lymphoma.
It has been proven that PUD is curable and rather easily. The cause of most PUD is infection with H. pylori. However, H. pylori infection is not routinely diagnosed, possibly because methods of testing for H. pylori infection are not satisfactory to physicians, especially the primary care physicians (i.e. invasive biopsy test). Therefore, primary physicians have tended to treat symptomatic patients with antisecretory agents.
Physicians need a simple, accurate and inexpensive diagnostic test for H. pylori infection so that they know when to treat patients and when to refer the patients to a gastroenterologist. However, the currently available H. pylori tests, which can be categorized as invasive tests and noninvasive tests, are not completely satisfactory.
The invasive tests require the use of endoscope followed by biopsy procedure. The tissue samples taken by the biopsy procedure can then be analyzed by culture, histology, or rapid urease testing.
Although culturing of the biopsy specimens provides the most reliable results for H. pylori testing, the reports of successful rates in a good laboratory are only between 70% and 80% (Han, S. W., et al., Eur. J. Clin. Microbiol. Infect. Dis. (1995), 14:349-352). Histological examination of special stained biopsy specimens can provide the direct evidence of acute or chronic inflammatory mucosal cells and lesions. However, it requires the collaborations of both an endoscopist and a pathologist (Genta, R. M., et al., Hum. Pathol. (1994), 25:221-226). Rapid urease tests detect the rise in pH from ammonia produced by H. pylori urease, which splits urea into ammonia and carbon dioxide. However, it requires a high density of bacteria and anything that reduces the bacterial load may produce a false-negative (Cutler, A. F., Am. J. Med. (1996), 100:35S-39S).
A number of noninvasive tests have been developed to detect the presence of H. pylori infection since 1990. For example, the Urea Breath Testing is based on the urease activity of the organism, which splits urea labeled with 13C or 14C into nonradioactive 13CO2 or radioactive 14CO2. The urea breath test is widely recommended for confirming eradication of H. pylori 4 weeks after therapy.
U.S. Pat. Nos. 5,716,791, 5,871,942, and 5,932,430 disclose immunoassays for detecting H. pylori antigens in stool specimens using a polyclonal antibody which is obtained from sensitizing animal with H. pylori cells (i.e., ATCC strain 43504). The antibody is purified by DEAE (diethylaminoethyl cellulose) column. Although the stool antigen test is reported to be satisfactory, the collection and process of the stool specimens are found to be difficult and unpleasant. Many patients are unwilling to provide stool samples to physician due to offensive odor and lack convenient collection device.
Serologic testing of serum H. pylori antibodies using ELISA is another widely used test. Examples of the latter techniques can be found in a U.S. Pat. No. 5,262,156 and EP Pat. No. 0 329 570. There have been several major antigens identified and used in immunoassays in the detection of H. pylori antibodies. However, these assays have not exhibited the specificity and sensitivity that are desired in serodiagnosis. (Newell, D. G., et al., Serodian. Immunother. Infec. Dis., (1989), 3:1-6). One of the problems derives from cross-reactivity. That is because the dominant antigens in H. pylori (e.g., the putative flagellar protein which has a molecular weight of 60 Da) are not specific to H. pylori. Some of these antigens can be found in other bacteria such as C. jeuni and C. coli. A second problem that has been encountered in designing immunoassays for H. pylori is strain variation. Substantial differences in the antigens have been observed in different strains of H. pylori. These problems preclude designing an assay around the use of a single antigen. One approach that has been taken to improving the specificity and selectivity of antibody immunoassays for H. pylori has been to use a mixture of antigens from different H. pylori strains which mixture is enriched with certain antigen fragments. One ELISA which detects H. pylori antibodies in blood sera is commercially available. This assay uses a bacterial whole cell lysate as the antigen.
There are other disadvantages of using an ELISA which employs antigens to detect the presence of H. pylori antibodies in serum. In particular, the antibody titer in human sera remains high for a prolonged time (in some cases as much as twelve months) after the infection has been treated. Consequently, a positive test using this ELISA does not necessarily mean that the patient is currently infected and requires treatment for H. pylori infection. When confronted with a positive ELISA, treating physicians often order a gastric biopsy to confirm the presence of the bacteria before initiating antibiotic therapy. Therefore, the antigen-based ELISA does not eliminate the need for the invasive procedure.
It is therefore the object of the present invention to design a noninvasive and highly accurate diagnostic test for H. pylori infection. During the course of the investigation, H. pylori antigens in blood are discovered, which are in the forms of DNA or fragments thereof, or proteins/peptides or other antigenic components thereof, exist in blood, including whole blood, plasma and serum. Special methods for detecting these H. pylori antigens are thus designed to provide evidence that antigenic fragments of H. pylori are existed in blood. These methods include, but not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR) and DNA hybridization for detecting nucleic acid fragments of H. pylori, using primers or oligonucleotides specific for H. pylori and/or DNA probes derived from H. pylori strains. Additionally, immunoassays and immunoblotting are also developed for detecting protein/peptide or any antigenic components of H. pylori, using an affinity purified antibody against H. pylori. 
As of this time, there has been no report with regard to the existence of H. pylori antigens in blood. The present invention will be the first to prove that H. pylori antigens not only exist in blood, but can be detected by the methods presented in the following sections.
The present invention provides H. pylori antigens which are existed in patient""s blood and can be detected by polymerase chain reaction (PCR), ligase chain reaction (LCR), DNA hybridization, RNA hybridization, branched DNA assay, immunoblotting, and immunoassay. The present invention also provides methods for diagnosing H. pylori infection by detecting the H. pylori antigens in blood.
The term xe2x80x9cantigensxe2x80x9d used in the present invention broadly covers any substances which are directly or indirectly capable, under appropriate conditions, of inducing a specific immune response and of reacting with the products of the response, that is, with specific antibody or specifically sensitized T-lymphocytes, or both. Examples of these substances include, but are not limited to, proteins/peptides, polysaccharides, lipids, and poly- or oligo-nucleotides.
There are particularly two special kinds of H. pylori antigens that can be detected in blood. The first kind relates to polynucleotides or oligonucleotides which are chromosomal DNA, RNA or fragments thereof from H. pylori. This kind of H. pylori can be detected by polymerase chain reaction (PCR), ligase chain reaction (LCR), and hybridization (preferably spotted DNA hybridization) methods or other amplification methods.
The PCR method provided in the present invention requires the use of a pair of primers specific for detecting H. pylori. The term xe2x80x9cprimerxe2x80x9d as used herein refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer product which is complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleotide triphosphates with appropriate enzymes at a suitable temperature. The term xe2x80x9coligonucleotidexe2x80x9d as used herein is defined as a molecule comprised of two or more deoxyribonucleotides and/or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be derived synthetically or by cloning.
The primers are prepared based upon conserved sequence found in consensus fragments of H. pylori strains, such as ATCC strains 43504, 43571, 43629, and 49053. The preferred primers range is from 15 to 25 base pairs (bps), most favorably about 20 bps in length. Better amplification can be obtained when both primers (forward and reverse primers) are the same length and with roughly the same nucleotide composition. The preferred blood sample for PCR is plasma.
The LCR method provided in the present invention requires the use of a DNA ligase and two sets of oligonucleotides which are specific to H. pylori. The preferred DNA ligase is Pfu DNA ligase, which is a thermostable DNA ligase isolated from Pyrococcus furiosus and is commercially available. The two sets of oligonucleotides for LCR is preferably longer in length than the primers for PCR. Like the PCR primers, the LCR oligonucleotides are derived from conserved sequence of the consensus fragments of H. pylori strains, such as ATCC strains 43504, 43571, 43629, and 49053.
LCR is performed by repeated cycles of heat denaturation of a DNA template containing a target sequence, annealing a first set of two adjacent oligonucleotide probes to the target DNA sequence in a unique manner, and a second set of complementary oligonucleotide probes that hybridize to the sequence opposite to the target DNA sequence. The term xe2x80x9ctarget sequencexe2x80x9d used herein refers to the xe2x80x9cchromosomal DNA or fragments thereofxe2x80x9d found in blood samples. Thereafter, the DNA ligase can covalently link each pair of adjacent probes provided there is complete complementary at the junction of the two adjacent probes.
The hybridization method requires the preparation of an H. pylori DNA probe. The H. pylori DNA probe is prepared by cutting out and extracting DNA fragment from H. pylori nucleic acid extracts after agarose gel electrophoresis. The probe normally has at least about 25 bases, more usually at least about 30 bases, and may have up to about 10,000 bases or more, usually having not more than about 5,000 bases. This DNA fragment is then digested with restriction endonucleases and ligated with a vector to form a recombinant plasmid construct, which can transfect eucaryotic or procaryotic host cells. The DNA fragment can be propagated in the host cells and re-isolated. The propagated DNA fragment can then be labeled with radioisotope (such as 32P, 3H, 14C, or the like) or fluorescence (such as the use of digoxigenin- and biotin-labeled DNA probes coupled with fluorescence detection methods) and used as a DNA probe.
The hybridization method is carried out by treating the nucleic acid sample from blood, preferably serum, with a denaturation agent to denature DNA on a solid phase support such as a nitrocellulose filter. The preferred denaturation agent include, but not limited to, alkali solution, elevated temperatures, organic reagents (e.g., alcohols, amides, amines, ureas, phenols and sulfoxides), or certain inorganic ions (e.g., thiocyanate and perchlorate). The labeled DNA probe will then be added to the denatured DNA spotted filter. The filter may then be assayed for the presence of DNA hybrids in the nature of the label. If the label is radioactive, the filter can be exposed to X-ray film. If the label is fluorescence, the filter can be viewed directly using a fluorescence microscope.
The second kind of antigens relates to H. pylori proteins and/or peptides, or any substances containing antigenic epitopes in blood which can be detected by immunoblotting or immunoassay, preferably using an affinity purified antibody against H. pylori antigens. Both primary and secondary antibodies may be required for detecting or measuring H. pylori antigens in blood, depending upon the kinds of methods used in the detection. A primary antibody is an antibody raised against an antigen, which in this case is the H. pylori antigen. The secondary antibody is an antibody against the immunoglobulin of a primary antibody producing species (such as goat anti-rabbit IgG). The preferred blood sample for the detection is serum.
Immunoblotting method is carried out by first analyzing blood sample with SDS-PAGE. After electrophoresis, the proteins/peptides bands are transferred to a nitrocellulose filter, which is then incubated with sufficient amount of anti-H. pylori antibody. An enzyme conjugated secondary antibody against the immunoglobulin of the animal species producing the anti-H. pylori can be added to the nitrocellulose filter. One of the preferred enzyme for this method is alkaline phosphatase, wherein the reaction can be detected by adding 5-bromo-4-chloro-3-indolylphosphate. Another preferred enzyme marker used in this method is horseradish peroxidase, which can be detected by adding 4-chloro-1-naphthol, tetramethylbenzidine, or 3,3xe2x80x2-diaminobenzidine to produce colored insoluble product for visualization.
The immunoassay methods comprise, but not limited to, basic sandwich assay, triple sandwich assay, and immunochromatographic assay.
In the basic sandwich assay, two primary antibodies are required, in which one is bound to a solid carrier and the other is labeled with a detection agent. The triple sandwich assay requires the combined use of two primary antibodies (namely, the first antibody and the second antibody) against H. pylori, in which only one primary antibody is required to be bound to a solid carrier, and one secondary antibody against the immunoglobulin of the animal species producing the unbounded primary antibody to form a complex against the antibody-antigen-antibody complex. The secondary antibody is labeled with a detection agent.
The solid carrier for the sandwich assays can be plastic beads, polyethylene, polystyrene, polypropylene, etc. The detection agent can be an enzymatic marker (such as alkaline phosphatase or horseradish peroxidase), a fluorescent or luminescent agent (such as fluorescein, rhodamine, or europium, luminol, or acridium), a radioisotope labeling (such as I125), or a color particle (such as gold, silver, blue-latex, or selenium).
Both the basic and triple sandwich immunoassays require the interaction of a first primary antibody against H. pylori to form an antigen-antibody complex, followed by contacting the antigen-antibody complex with a second antibody against H. pylori. 
The immunochromatographic assay also requires the combined use of the two primary antibodies against H. pylori. Contrasting to the basic and triple sandwich assays, the first antibody (i.e., the antibody which is in touch with the biological specimen first) is labeled with color particles. The second antibody is bound to a solid carrier such as nitrocellulose (or nitrocellulose derivative) membrane, nylon membrane, polyester membrane, filter paper, agarose or sephedex gel. The preferred solid carrier for the immunochromatographic assay is the nitrocellulose membrane. Optionally, a secondary antibody against the animal species for producing the first antibody can be added and/or bound to the solid carrier at near the end of the chromatographic strip opposite to the sample addition site. This secondary antibody is used as a control for capturing the unbound color particles at the end of the chromatographic run. Thus, if the sample does not contain H. pylori antigens, the color particles labeled first antibody will run through the second antibody without binding to it because no antibody-antigen-antibody complex is formed. However, because the secondary antibody is against the immunoglobulin of the first antibody producing animal, it will bind to the first antibody when it runs by regardless whether the first antibody has form a complex with the sample. The binding between the first antibody and the secondary antibody shows the end of the immunochromatographic run.
One of the problems in dealing with serum sample is that a patient infected with H. pylori often carries with him/her H. pylori antibodies in the serum. These serum H. pylori antibodies can form immune complexes with serum H. pylori antigens which may have impact on the accuracy of the immunoassays of the present invention. The ways to dissociate the H. pylori immune complexes include, but not limited to, dissociating the complexes with a dissociation reagent or at a sample dissociation condition.
Examples of the dissociation reagent include, but not limited to, high salts (e.g., 0.2 M to 1.5 M of 1 M NaCl, or KCl (most preferably, 1 M of NaCl or KCl), detergents (e.g., 0.1 to 2.0% (most preferably 1%) of sodium dodecyl sulphate (SDS), 0.1 to 2.0% (most preferably 1%) of TWEEN 20, 0.1 to 2.0% (most preferably 1%) of octylglucoside, 0.1 to 2.0% (most preferably 1%) of deoxycholate, or 0.1 to 2.0% (most preferably 1%) of TRITON X-100), chaotropic agents (e.g., 0.5 M to 6 M of guanidine HCl, 0.5 M to 8 M of urea, or 0.5 M to 3 M of KSCN), organic solvents (e.g., 10% dioxane or 40% ethylene glycol), enzymes (e.g., 1 to 10 units/ml of proteases (such as trypsin, chymotrypsin, pepsin, V8 protease, and subtilisin) or 1 to 10 units/ml of lipases (such as lipoprotein lipase from bovine milk, and lipase from Candida rugosa)). After the completion of the dissociation, the dissociation reagent can be removed from the serum samples by conventional methods such as dilution, filtration, column chromatography, or dialysis.
Examples of sample dissociation condition include, but not limited to, high pH (e.g., pH xe2x89xa79) or low pH (e.g., pH xe2x89xa63), and/or elevated temperature (e.g., at least 50xc2x0 C.). After the completion of the dissociation, the condition of the serum sample can be re-adjusted back to the original pH (i e., at pH 7.4) or temperature (i e., at room temperature) by conventional methods.
Furthermore, the dissociation treated serum sample can be treated with a protein based reagent to minimize cross-reactivity. The preferred protein based reagent contains at least one of the following proteins: fetal bovine serum, pig serum, normal goat serum, horse serum, casein, albumin, gelatin, and bovine serum albumin.